A variety of small metabolites have been found to regulate gene expression in bacteria, fungi and plants via direct interactions with distinct mRNA folds. {Breaker, R R 2011; Garst A D et al 2011; Deigan K E & Ferré-D'Amaré A R 2011; Serganov A & Patel D J 2012}
In this form of regulation, the target mRNA typically undergoes a structural change in response to metabolite binding. {Blouin S et al 2009; Nudler E & Mironov A S 2004; Schwalbe H et al 2007; Serganov A & Nudler E. 2013; Fiegland, L. R., et al 2012}
These mRNA elements have thus been termed “riboswitches”, and generally include both a metabolite-sensitive aptamer sub-domain and a so-called expression platform. For riboswitches that regulate the process of translation, the expression platform minimally consists of a ribosomal recognition site (Shine-Dalgarno S D). In their simplest form, the SD sequence is found to overlap with the metabolite-sensitive aptamer domain at its downstream end Representative examples include the S-adenosylmethionine class II (SAM-II) and the S-adenosylhomocysteine (SAH) riboswitches, as well as pre-queuosine class I (preQ1-I) and II (preQ1-II) riboswitches. {Roth A et al 2007; Meyer M M et al 2008}
The secondary structures of these four short RNA families contain a pseudoknot fold which is central to their gene regulation capacity. While the SAM-II and preQ1-I riboswitches fold into classical pseudoknots {Gilbert S D et al 2008; Klein D J et al 2009}, the conformations of the SAH and preQ1-II counterparts are more complex and include a structural extension that contributes to the pseudoknot architecture. {Meyer M M et al 2008.}
Importantly, the impact and evolutionary importance of the “extra” stem-loops on the function of the SAH and preQ1-II riboswitches remain unclear.
PreQ1 riboswitches interact with the bacterial metabolite 7-aminomethyl-7-deazaguanine, a precursor molecule in the biosynthetic pathway of queuosine, a modified base encountered at the wobble position of some transfer RNAs. {Meyer M M et al 2008} The general biological significance of studying the preQ1-II system stems from the fact that this gene-regulatory element is found almost exclusively in the Streptococcaceae bacterial family and that the preQ1 metabolite is not generated in humans but has to be acquired from the environment. {Meyer M M et al 2008.} Correspondingly, the preQ1-II riboswitch represents a putative target for antibiotic interventions. Class I preQ1 riboswitches have been extensively investigated. {Liberman, J. A. & Wedekind, J. E. 2011; Kang, M., et al 2009; Spitale, R. C., et al 2009; Rieder, U., et al 2009; Rieder, U., et al 2010; Feng, J., et al 2011; Zhang, Q., et al 2011; Eichhorn, C. D., et al 2012; Jenkins, J. L., et al 2011; Santner, T., et al 2012; Yu, C.-H., et la 2013.} However, class II riboswitches have been largely overlooked despite the fact that a different mode of ligand binding has been postulated. {Meyer M M et al 2008} Consequently, the molecular details of the preQ1 ligand-RNA interaction are currently not known and high-resolution structures of both free and ligand-bound class-II aptamers are lacking.
The consensus sequence and the secondary structure model for the preQ1-II motif (COG4708 RNA) is shown in FIG. 1a and comprises approximately 80 to 100 nucleotides. {Meyer M M et al 2008} The minimal Streptococcus pneumoniae R6 aptamer domain sequence binds preQ1 with submicromolar affinity and consists of an RNA segment forming two stem-loops, P2 and P3, and a pseudoknot PS (FIG. 1b). In-line probing studies suggest that the putative Shine-Dalgarno (SD) box (AGGAGA, FIG. 1) is sequestered by pseudoknot formation, which would result in translational-dependent gene regulation of the downstream gene. {Meyer M M et al 2008}
Here, we investigated folding and ligand recognition of the S. pneumoniae R6 preQ1 class II riboswitch, using complementary chemical, biochemical and biophysical methods including selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), mutational analysis experiments, 2-aminopurine fluorescence, and single-molecule fluorescence resonance energy transfer (smFRET) imaging. In so doing, we explored the functional impact of the additional stem-loop element in an otherwise “classical” pseudoknot fold of the preQ1-II riboswitch on the dynamics of pseudoknot folding. Our results reveal that the unique 3′-stem-loop element in the preQ1-II riboswitch contributes to the process of SD sequestration, and thus the regulation of gene expression, by modulating both its intrinsic dynamics and its responsiveness to ligand binding.
We also have investigated the thiamine pyrophosphate(TPP)-sensing riboswitch, which is one of the earliest discovered regulatory elements in mRNA that is prevalent among bacteria, archaea, fungi and plants (10-12). {Winkler W, et al 2002); Sudarsan N, et al 2003; Cheah M T, et al 2007}
TPP riboswitches, sometimes present in tandem, control genes that are involved in the transport or synthesis of thiamine and its phosphorylated derivatives. {Sudarsan N, et al 2006; Welz R, Breaker R R 2007} The TPP-bound aptamer adopts a uniquely folded structure in which one sensor helix arm (P2/P3) forms an intercalation pocket for the pyrimidine moiety of TPP, while the other sensor helix arm (P4/P5) offers a water-lined binding pocket for the pyrophosphate moiety of TPP that engages bivalent metal ions (FIG. 12). {Serganov A, et al 2006; Thore S, et al 2006; Edwards T E, et al 2006} Like most riboswitch domains, structural information pertaining to the ligand-free TPP riboswitch is relatively lacking. Consequently, little is presently known about the determinants of alternative riboswitch folding pathways and how ligands regulate these events. A deeper understanding of both bound and unbound forms of the aptamer and expression platforms is required to gain mechanistic insights into the regulatory switch that they induce. {Haller A, et al 2011; Liberman J A & Wedekind J E 2011; Perdrizet G A II, et al 2012; Wong T N & Pan T 2009} The nature and timing of the folding-recognition process in riboswitches implies potentially complex and rapid dynamic processes within the nascent RNA chain. {Al-Hashimi H M & Walter N G 2008} Knowledge of these events is therefore critical to achieving a complete understanding of riboswitch-mediated gene regulation.
Previous investigations have employed a battery of distinct biophysical methods to explore the nature of the TPP ligand recognition process. Such studies include 2-aminopurine fold analysis (2Apfold) {Lang K, et al 2007}, small-angle X-ray scattering (SAXS) {Ali M, et al 2010; Baird N J, et al 2010; Baird N J & Ferré-D'Amaré A R 2010}, RNase-detected selective 2′-hydroxyl acylation (SHAPE) {Steen K-A, et al 2010; Steen K-A, et al 2012}, isothermal titration calorimetry (ITC) {Kulshina N, et al 2009; Burnouf D, et al 2012}, as well as single-molecule optical-trapping methods in which force was applied via the 5′ and 3′ ends of the RNA to directly monitor the energy landscape of TPP riboswitch folding and unfolding. {Anthony P C, et al 2012}
Investigations of this kind have provided an important framework for understanding global features of the TPP riboswitch aptamer domain, revealing that its structural compaction is enabled by physiological concentrations of Mg2+ ions and enforced by TPP binding. Two additional, generally agreed upon, features of the TPP riboswitch have been derived from these experiments. First, secondary structures of the P2/P3 and P4/P5 ligand sensor arms form in the presence of Mg2+ alone. Second, tertiary interactions between the two sensor arms (e.g. P3/L5) do not form in the absence of TPP binding. However, it is not yet clear how the collapse of the two helical domains and formation of these tertiary interactions are influenced by ligand binding or whether they are essential to binding pocket formation. Here, we have employed single-molecule fluorescence resonance energy transfer (smFRET) imaging {Roy R, et al 2008; Lemay J F, et al 2006; Wood S, et al 2012} to track ligand-dependent changes in the TPP riboswitch from multiple structural perspectives in order to elucidate the relationship between TPP recognition and aptamer folding